H. Allen O. Hill

The immobilisation of proteins in carbon nanotubes

Carbon nanotubes, fullerene-related structures, have been used for the immobilisation of proteins and enzymes. We have been able to demonstrate, for the first time, direct imaging by high resolution transmission electron microscopy of Zn2Cd5-metallothionein, cytochromes c, c, and β-lactamase 1. This was achieved, without modification, because the biomolecules encapsulated within nanotubes appear to be shielded from the consequences of exposure to the intense electron beam. The results indicate that the internal surface of the nanotubes interacts strongly with the enzymes resulting in their immobilisation. In some cases, the proteines are seen to be distorted giving a concave meniscus inside the tubes. Single protein molecules, their dimers, tetramers and higher oligomers are observed inside the central cavity. Comparison of the catalytic activities of immobilised β-lactamase 1 on or in nanotubes with the free enzyme in the hydrolysis of penicillin, however, showed a significant amount of the immobilised enzyme remained catalytically active, implying that no drastic conformational change had taken place. The carbon nanotube appears to act as a benign host in its ability to encapsulate protein molecules within an environment which offers some protection.

[1] Chemistry of dioxygen

Publisher Summary The chemistry of molecular oxygen, or dioxygen as it is increasingly coming to be called, is dominated by the relative reluctance with which the element reacts with most, but not all, compounds. This quality is rarely thermodynamic in origin; rather the slow rate of reaction is associated with either the strong oxygen–oxygen bond or the character of the ground state of dioxygen or both. If a reaction is to take place with dioxygen in its ground state, there must be a change of spin at some stage during the reaction. This is forbidden, within the limitations of the descriptions used; at the very least, the reactions are improbable. For dioxygen to react rapidly, this spin restriction should be removed or the oxygen–oxygen bond should be partially weakened or both of these should occur simultaneously. It is not surprising that much of the chemistry of dioxygen is concerned with reactions with paramagnetic species, with electron-donating species, with light, or with various combinations of these three factors. This chapter discusses the chemistry of the reduction products of dioxygen, but one should be aware of the marked influence of all of these factors on most reactions of dioxygen. They are often, if not always, responsible for the promotion or catalysis of oxidation reactions.

Metal ions and complexes as modulators of protein-interfacial electron transport at graphite electrodes

Abstract An extensive investigation of the direct (unmediated) electrochemical activity of various redox proteins at pyrolytic graphite electrodes has been undertaken. With the exception of the “blue” copper protein azurin, a profound preference for the hydrophilic “edge” over the hydrophobic “basal” plane orientation of the graphite surface is observed. This may be identified with the presence of various oxidised (CO) functionalities at the polished “edge” surface which, most probably in a random manner, constitute reversible and productive binding domains for the proteins. Conditions under which the rates and reversibility of heterogenous electron transport may be optimised depend upon the protein under examination. Well-behaved electrochemistry, indicate of diffusion-dominated heterogeneous electron transport, is modulated by electrode surface protonation (pK = 5.6) and levels of redox-inert multivalent cations, including Mg2+ and Cr(NH3)3+6. The electrochemistry of several proteins which have negatively charged interaction domains, including plastocyanin, and chloroplast and bacterial ferrodoxins, is promoted and stabilised by electrode surface protonation, and interfacial binding of multivalent cations which is attenuated at high ionic strength. Coversely, the electrochemistry of horse-heart cytochrome c, for which the region around the exposed heme edge carries a net positive charge, is inhibited by electrode surface protonation and destablished by the presence of multivalent cations. These patterns of behaviour may be rationalised in terms of a heterogeneous electrode surface which comprises regions of hydrophilic polar groups at which proteins may associate reversibly if resultant coulombic interactions are favourable, and regions of extensive hydrophobicity at which less reversible and (probably) degradative adsorption occurs. Within this basic model, there is considerable scope for domain selectivity which may arise from variations in medium and short range order and distribution of CO functionalities. Implications for the control of in vivo electron-transport processes are discussed.

The production of hydroxyl and superoxide radicals by stimulated human neutrophils- measurements by EPR spectroscopy.

Neutrophilic polymorphonuclear leukocytes (neutrophils) consume considerably more oxygen in association with phagocytosis than in the resting state. This extra respiration of phagocytosis [l] is not affected by inhibitors of mitochondrial electron transport and is distinct from the process of phagocytosis itself, which proceeds [2] normally under anaerobic conditions. The oxidase system appears to involve a novel cytochrome b [3,4] and is important [5] for the microbicidal processes responsible for the killing of certain bacteria. II I In

Ferricinium ion as an electron acceptor for oxido-reductases

Abstract The ability of ferrocene monocarboxylic acid to act as a mediator to some oxido-reductases was investigated. Dc cyclic voltammetry was used to study the coupling of the electrode reaction, associated with the oxidation of ferrocene to ferricinium ion, to an enzymatic oxidation reaction. The reactions catalysed by pyruvate oxidase, xanthine oxidase, sarcosine oxidase, lipoamide dehydrogenase, glutathione reductase and alcohol dehydrogenase were investigated. The rates of reaction between the ferricinium ion and the reduced form of each of the enzymes, in the presence of their substrates, were measured.

A method for estimation of hydrogen peroxide based on mediated electron transfer reactions of peroxidases at electrodes

Abstract The development of a sensitive electrochemical assay for low levels of hydrogen peroxide is described. The system is based on the enzymic reduction of substrate (H2O2) by peroxidase and subsequent electron transfer from a gold or pyrolytic graphite electrode to the enzyme, via a redox mediator. Cyclic voltammetry has been employed to assess the ability of certain redox-active couples to act as mediator. A linear response to hydrogen peroxide is observed in the concentration range 5 × 10−8 –6 × 10−2M.

Bioelectrochemical fuel cell and sensor based on a quinoprotein, alcohol dehydrogenase

Abstract A biofuel cell, yielding a stable and continuous low-power output, based on the enzymatic oxidation of methanol to formic acid has been designed and investigated. The homogeneous kinetics of the electrochemically-coupled enzymatic oxidation reaction were investigated and optimized. The biofuel cell also functioned as a sensitive method for the detection of primary alcohols. A method for medium-scale preparation of the enzyme alcohol dehydrogenase [alcohol:(acceptor) oxidoreductase, EC 1.1.99.8] is described.

Novel method for the investigation of the electrochemistry of metalloproteins: cytochrome c

The d.c. and a.c. cyclic voltammetries of horse heart ferricytochrome c have been investigated and it is shown that in the presence of 4,4′-bipyridyl, the electrochemistry corresponds to a quasi-reversible one-electron process, from which an E° value of +0·25 V vs. normal hydrogen electrode can be derived.

Surface-modified mutants of cytochrome P450cam: enzymatic properties and electrochemistry

We report the electrochemistry of genetic variants of the haem monooxygenase cytochrome P450cam. A surface cysteine‐free mutant (abbreviated as SCF) was prepared in which the five surface cysteine residues Cys‐58, Cys‐85, Cys‐136, Cys‐148 and Cys‐334 were changed to alanines. Four single surface cysteine mutants with an additional mutation, R72C, R112C, K344C or R364C, were also prepared. The haem spin‐state equilibria, NADH turnover rates and camphor‐hydroxylation properties, as well as the electrochemistry of these mutants are reported. The coupling of a redox‐active label, N‐ferrocenylmaleimide, to the single surface cysteine mutant SCF‐K344C, and the electrochemistry of this modified mutant are also described.

Investigation of the binding of inorganic complexes to blue copper proteins by 1H nmr spectroscopy, I. The interaction between the [Cr(phen)3]3+ and [Cr(CN)6]3− ions and the copper(I) form of parsley plastocyanin

Abstract The effects of the addition of [Cr(phen) 3 ](ClO 4 ) 3 and K 3 [Cr(CN) 6 ] on the 1 H nmr spectrum of the copper(I) form of parsley plastocyanin are described. It is concluded that the ions [Cr(phen) 3 ] 3+ and [Cr(CN) 6 ] 3− bind to different parts of the protein.